1. Field of the Invention
The invention relates to genetic and cellular factors associated with cellular senescence and immortalization, apoptosis, neoplastic transformation and sensitivity to chemical and physical environmental insult. More particularly, the invention also provides methods and reagents for activating a particular cellular factor, the cellular tumor suppressor known as p53, and specifically provides reagents for activating p53-dependent transcriptional activity. The reagents provided by the invention comprise recombinant expression constructs encoding all or a portion of a particular cellular gene, termed ING1, and the protein produced by expression of this gene, known as p33.sup.ING1. The invention provides methods for using such recombinant expression constructs for activation of p53-related transcription involved in expression of apoptosis and other cellular expression pathways. The invention also provides reagents and methods for preparing genetic suppressor elements (GSEs) from ING1-encoding nucleic acid species, and methods for using such GSE for inhibiting apoptosis, delaying cellular aging, facilitating anchorage-independent growth, protecting the cell from the effects of certain cytotoxic drugs, and inhibiting the activity of p53. This invention also provides methods for characterizing mammalian cells on the basis of whether such cells express the ING1 gene, and embodiments of such methods directed at malignant or pre-malignant tissues in an animal for assaying the risk of developing malignant disease by the animal.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United States. Clinically, a broad variety of medical approaches, including surgery, radiation therapy and chemotherapeutic drug therapy are currently being used in the treatment of human cancer (see the textbook CANCER: Principles & Practice of Oncology, 2d Edition, De Vita et al., eds., J. B. Lippincott Company, Philadelphia, Pa., 1985). However, it is recognized that such approaches continue to be limited by a fundamental lack of a clear understanding of the precise cellular bases of malignant transformation and neoplastic growth.
The beginnings of such an understanding of the cellular basis of malignant transformation and neoplastic growth have been elucidated over the last ten years. Growth of normal cells is now understood to be regulated by a balance of growth-promoting and growth-inhibiting genes, known as proto-oncogenes and tumor suppressor genes, respectively. Proto-oncogenes are turned into oncogenes by regulatory or structural mutations that increase their ability to stimulate uncontrolled cell growth (see Varmus, 1989, "A historical overview of oncogenes", in Oncogenes and the Molecular Origin of Cancer, Weinberg, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 3-44).
It is likely, however, that there are at least as many cancer-associated genes that are involved in suppression rather than induction of abnormal cell growth. This class of genes, known as anti-oncogenes or tumor suppressors, has been defined as comprising "genetic elements whose loss or inactivation allows a cell to display one or another phenotype of neoplastic growth deregulation" by Weinberg (1991, Science 254: 1138-1146). Changes in a tumor suppressor gene that result in the loss of its function or expression are recessive, because they have no phenotypic consequences in the presence of the normal allele of the same gene. The recessive nature of mutations associated with tumor suppressors makes such genes very difficult to analyze or identify by gene transfer techniques and explains why oncogene research is far more advanced than studies of tumor suppressors.
In normal cells, tumor suppressor genes may participate in growth inhibition at different levels, from the recognition of a growth inhibiting signal and its transmission to the nucleus, to the induction (or inhibition) of secondary response genes that finally determine the cellular response to the signal. The known tumor suppressor genes have indeed been associated with different steps of the regulatory pathway. Thus, the DCC and ErbA genes encode receptors of two different classes (Fearon et al., 1990, Science 247: 49-56; Sap et al., 1986, Nature 324: 635-640; Weinberger et al., 1986, Nature 324: 641-646). The gene NF-1 encodes a polypeptide that resembles ras-interacting proteins, that are members of the signaling pathway (Xu et al., 1990, Cell 62: 599-608; Ballester et al., 1990, Cell 62: 851-859; Buchberg et al., 1990, Nature 347: 291-294; Barbacid, 1987, Ann. Rev. Biochem. 56: 779-827). p53, RB and WT genes encode nuclear regulatory proteins (Fields et al., 1990, Science 249: 1046-1049; Raycroft et al., 1990, Science 249: 1049-1051; Kern et al., 1991, Oncogene 6: 131-136; O'Rourke et al., 1990, Oncogene 5: 1829-1832; Kern et al., 1991, Science 252: 1708-1711; Lee et al., 1987, Nature 329: 642-645; Friend et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9059-9063; Call et al., 1990, Cell 60: 509-520; Gessler et al., 1990, Nature 343: 774-778).
Two approaches have been previously used for cloning tumor suppressor genes. The first approach is based on isolating the regions associated with nonrandom genetic deletions or rearrangements observed in certain types of tumors. This approach requires the use of extremely laborious linkage analyses and does not give any direct information concerning the function of the putative suppressor gene (Friend et al., 1991, Science 251: 1366-1370; Viskochil et al., 1990, Cell 62: 187-192; Vogelstein et al., 1988, N. Engl. J. Med. 319: 525-532). In fact, among numerous observations of loss of heterozygosity in certain tumors (Solomon et al., 1991, Science 254: 1153-1160; LaForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88: 5036-5040; Trent et al., 1989, Cancer Res. 49: 420-423), there are only a few examples where the function of the affected gene is understood. In two of these rare cases the gene function was identified using another method, analysis of dominant negative mutant proteins (Herskowitz, 1987, Nature 329: 219-222).
Specifically, the tumor suppressor gene p53 was first discovered as an altered forms which encoded a mutant protein (Sap et al., 1986. ibid.; Weinberger et al., 1986, ibid.; Raycroft et al., 1990, ibid.; Milner et al., 1991, Molec. Cell. Biol. 11: 12-19). p53 is a tumor suppressor gene that mediates cell response to various types of stress leading the cell growth arrest, apoptosis or other responses (i.e. differentiation) through modulation of expression of p53-responsive genes. p53 pathway is inactivated in the majority of human cancers making its restoration a major goal of gene therapy. At the same time, p53 pathway determines sensitivity of several tissues to DNA damaging treatments, including chemotherapy and gamma radiation. Therefore, stimulation or restoration of the p53 pathway could be critically important for the efficacy of anti-cancer therapy, while suppression of p53 pathway could be used to defend sensitive tissues from genotoxic stress and for the generation of immortal cell lines also requiring p53 functional inactivation. Oncogenic mutant p53 protein forms functionally inactive complexes with the wild-type protein; such complexes fail to provide the normal negative regulatory function of the p53 protein (Herskowitz, 1986, ibid.; Milner et al., 1991, ibid.; Montenarh & Quaiser, 1989, Oncogene 4: 379-382; Finlay et al., 1988, Molec. Cell. Biol. 8: 531-539).
The discovery and analysis of new recessive genes involved in neoplastic transformation has been greatly accelerated through the use of genetic suppressor elements (GSEs), derived from such genes and capable of selectively suppressing their function. GSEs are dominant negative factors that confer the recessive-type phenotype for the gene to which the particular GSE corresponds. Recently, some developments have been made in the difficult area of isolating recessive genes using GSE technology. Roninson et al., U.S. Pat. No. 5,217,889 (issued Jun. 8, 1993) teach a generalized method for obtaining GSEs (see also Holzmayer et al., 1992, Nucleic Acids Res. 20: 711-717). Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90: 3231-3235 teach isolation of GSEs from topoisomerase II cDNA that induce resistance to topoisomerase II-interactive drugs. Co-pending U.S. patent application Ser. No. 08/204,740, filed Mar. 2, 1994 disclosed the use of GSE technology to isolate neoplastic transformation-specific GSEs that were found to be derived from a variety of cellular genes previously unsuspected of any role in neoplastic transformation.
More recently, the GSE technology has been used to isolate and identify a new human tumor suppressor gene, termed ING1, that encodes a protein product termed p33.sup.ING1 (see International Application, Publication No. WO97/21809, published Jun. 19, 1997). The ING1 genc encodes a nuclear protein p33.sup.ING1, overexpression of which inhibits growth of different cell lines. The ING1 gene encodes a ubiquitously expressed, nucleus-localized zinc-finger protein that is highly conserved in evolution. Inhibition of ING1 gene expression, for example, by antisense RNA promotes anchorage independent growth in mouse breast epithelial cells and in Ela+ras-transformed mouse fibroblasts, while ING1 gene overexpression leads to G1 growth arrest in several cell lines tested. These properties (and others) of ING1 suggest its involvement in negative regulation of cell proliferation and in control of cellular aging, anchorage dependence and apoptosis (Garkavstev et al., 1996, Nature Genetics 14: 415-420; Garkavstev et al., 1997, Mol Cell Biol. 17: 2014-2019; Helbing et al., 1997, Cancer Res. 57: 1255-1258). This protein has been shown to be involved in negative regulation of mammalian cell proliferation and control of cellular aging, apoptosis and anchorage-dependent growth. These cellular functions have been recognized to depend largely on the activity of the p53 tumor suppressor gene described above (Gottlieb & Oren, 1996, Biochim. Biophys. Acta Rev. Cancer 1287: 77-102).
In addition, ING1 has been mapped to human chromosome 13q34 (Garkavstev et al., 1997, Cytogenet. Cell Genet. 76: 176-178; Zeremski et al., 1997, Somatic Cell Mol. Genet. 23: 233-236), and loss of heterozygosity in this chromosomal locus has been reported in squamous cell carcinomas of head and neck (Maestro et al., 1996, Cancer Res. 56: 1146-1150). Limited analysis of tumor cell lines revealed mutation of ING1 in neuroblastoma cell line and reduced expression in several breast carcinoma cell lines (Garkavstev et al., 1996, Nature Genetics 14: 415-420).
There remains a need in the art to characterize mammalian tumor suppressor genes to better understand the interrelatedness of the role(s) played by these genes in control of cell growth. More specifically, there is a need in the art to determine the interrelatedness of p33 and p53 in mediating their effects on control of cell growth and aging. In particular, there is a need in the art to understand how manipulation of p33.sup.ING1 and/or p53 gene expression, using GSE- and recombinant DNA technologies, can be used to promote or inhibit cell growth where appropriate. There is a great need in the art to determine whether expression or lack of expression of p33.sup.ING1 is related to neoplastic transformation and malignant disease, as has been shown for p53. Finally, there is a need in the art to establish whether a more complete assessment of a human patient for the risk of developing malignant or proliferative disease requires a determination of the status of both p53 and ING gene expression.